Administration of products of the 5-lipoxygenase metabolic pathway to enhance antimicrobial defense

ABSTRACT

The use of leukotrienes and other products of the 5-lipoxygenase pathway to enhance bacterial defense and treat infections is described. The products are especially useful when administered to the lungs for the treatment of pneumonia and other lower respiratory tract infections. The products may be administered for treatment or prophylactic purposes and may be administered concomitantly with antibiotics to combat infection.

FIELD OF THE INVENTION

The present invention relates generally to the administration ofcompounds to enhance antimicrobial defense, and more particularly to theadministration of products of the 5-lipoxygenase metabolic pathway toenhance bacterial defense and to treat and prevent bacterial pneumonia.

This invention was made with United States government support awarded bythe National Institute of Health (NIH), Grant Nos. HL 58200, HL 57243,AA 10571, P 50 HL 46487, CA 66180, HL 50057, and HL 47391.

BACKGROUND OF THE INVENTION

A. Pulmonary Host Defense and the Pathogenesis of Pneumonia

In view of the constant challenge to the respiratory tract from inhaledor aspirated microbes, and the deleterious consequences of uncheckedinfection, an efficient system of pulmonary antimicrobial defense isobviously important to health. Microbes which elude the mechanicaldefenses offered by the upper respiratory tract and airways reach thealveoli. Here, the alveolar macrophage normally serves as the defenderof mucosal sterility, patrolling the alveolar surface and clearingorganisms by phagocytosis and intracellular killing. M. Lohmann-Mattheset al., Eur. Respir. J. 7:1678-1689 (1994)!. If the microbial loadexceeds the local clearance capacity of the resident alveolarmacrophages, the macrophages can elaborate chemotactic factors whichrecruit circulating neutrophils to the airspaces and activate theirphagocytic and microbicidal activities.

Although phagocytic cells are capable by themselves of microbialingestion, the efficiency of this process is enhanced by the presence ofvarious soluble molecules (opsonins) which coat the organisms andmediate their attachment to surface receptors on the phagocyte. Theseopsonins include immunoglobin as well as factors which coat microbesnonspecifically, such as the complement fragment C3b, surfactant proteinA, and fibronectin. Likewise, phagocytosis and intracellular killing arefurther augmented by a variety of activating agents, including colonystimulating factors, chemokines, and lipids. Unfortunately, qualitativeor quantitative impairment of any component of these defenses cancompromise bacterial clearance and predispose to pneumonia. See, e.g., JLangermans et al., J. Immunol. Methods 174:185-194 (1994)!.

B. Significance of Bacterial Pneumonia

In industrialized nations, pneumonia is associated with greatermorbidity and mortality than any other type of infection. Overall, it isthe sixth leading cause of death in the United States. In adults greaterthan 65 years of age, it is the fourth most common reason forhospitalization. Among hospital-acquired infections, pneumonia is thesecond most common in incidence and the most commonly fatal.

Bacteria are the etiologic pathogens in a substantial proportion ofcommunity-acquired pneumonias and in the great majority of nosocomialpneumonias. Frequently, enteric Gram-negative organisms are theetiologic microbes responsible for both types of pneumonia.Gram-negative pneumonias are generally thought to result frommicroaspiration of oral secretions, and are therefore particularlylikely in individuals demonstrating oropharyngeal colonization withthese organisms. This is especially common in hospitalized patients,particularly those in intensive care units, but also occurs inalcoholics, patients with underlying systemic illness or impairments inhost defense, and those with chronic pulmonary disease. S. Nelson etal., Clin. Chest Med. 16:1-12 (1995)!.

C. Antibiotic Therapy

Due to the widespread use and frequent over-prescribing of antimicrobialagents, there is an increasing incidence of microbes acquiringdrug-resistance. In other words, organisms typically susceptible (i.e.,inhibited or killed) to a particular antimicrobial agent are no longersusceptible. This is especially important with regard to the use ofantibiotics in the treatment of bacterial infections.

Acquired drug resistance is usually caused by a mutation within thegenome of the microbe or by the acquisition of a plasmid. For example,one of the major mechanisms of resistance to the β-lactam antibiotics,including penicillins, is the production of β-lactamases. Moreover,resistance to one member of a class of agents (e.g., the aminopenicillinampicillin) can result in complete cross-resistance to other members ofthat class (e.g., the aminopenicillin amoxicillin).

Antibiotic pressure in certain patient populations (e.g., patients withunderlying systemic illness or impairments in host defense) hascontributed to the development of infections with multi-drug resistantorganisms, the eradication of which is increasingly difficult. Onefactor contributing to antibiotic pressure is the widespread use ofantibiotics in the hospital setting, especially in the critical careunits. Indeed, physicians are frequently forced to utilize antibioticregimens comprising multiple agents to combat such infections or to usebroad-spectrum agents (e.g., Primaxin®, Merck) generally reserved forthe most serious infections.

What is needed is a means for enhancing pulmonary defense capabilitiesthat either requires no antibiotics or can be used to augment antibiotictreatment. The enhancement means should be efficacious in the treatmentand prevention of bacterial pneumonia in those patients who areespecially susceptible thereto, should have a rapid onset of action, andshould not elicit immunological reactions in the recipient.

SUMMARY OF THE INVENTION

Leukotrienes are potent mediators of inflammation derived from the5-lipoxygenase pathway of arachidonic acid metabolism. These substanceshave been implicated in the pathogenesis of inflammatory lung diseases,and new pharmacologic agents that inhibit leukotriene synthesis oractions have recently become available for the treatment of asthma. Thepresent invention contemplates the use of leukotrienes and otherproducts of the 5-lipoxygenase pathway as an adjunct in the treatment ofpneumonia and other lower respiratory tract infections.

In order to evaluate the role of leukotrienes in bacterial pneumonia,the present inventors have employed a model of Klebsiella pneumonia inknockout mice rendered leukotriene-deficient by the targeted disruptionof the 5-lipoxygenase gene. The present inventors found that leukotrieneproduction was increased in the lungs of infected wild type mice, andthat leukotriene-deficient animals manifested reduced bacterialclearance and enhanced lethality. Moreover, alveolar macrophages fromknockout mice exhibited impaired in vitro phagocytosis and killing of K.pneumoniae, and this functional defect in leukotriene-deficient alveolarmacrophages was overcome by the addition of exogenous leukotrienes suchas LTB₄. Importantly, intrapulmonary administration of LTB₄ partiallyovercame the in vivo impairment in bacterial clearance observed inknockout mice.

The present inventors have determined that endogenous leukotrienes playan integral role in the host response to pulmonary infection. Even moreimportantly from a therapeutic standpoint, the present inventors foundthat exogenous leukotrienes exert pharmacologic actions which augmentthis response.

The present invention contemplates the treatment of patients with arecognized predisposing factor (e.g., smoking, alcoholism, diabetes, HIVinfection, known aspiration) for overwhelming pneumonia, or with earlypneumonia, with administration via inhalation or an endotracheal tube ofmetabolic products of the 5-lipoxygenase pathway (e.g., leukotrienes).In addition, the present invention contemplates the use of the productsof the 5-lipoxygenase pathway for prophylactic purposes. While anunderstanding of the mechanism by which the products act is notnecessary for the successful practice of the present invention, theadministration of such products, especially the intrapulmonaryadministration of leukotrienes, augments local endogenous host defensemechanisms and assists in bacterial infection eradication duringantibiotic administration. The products have a relatively short durationof action (e.g., hours), do not cause antibody-mediated immuneresponses, and are relatively inexpensive.

The present invention is not limited to the intra-pulmonaryadministration of products of the 5-lipoxygenase pathway for thetreatment of pneumonia. Indeed, the present invention contemplates theadministration of these products via other routes of administration andfor the treatment and prevention of other conditions. The products maybe administered concomitantly with antibiotics in some embodiments. Inother embodiments, different products (e.g., LTB₄ and LTC₄) of the5-lipoxygenase pathway are administered together or at definedintervals, with or without the concomitant administration ofantibiotics.

The present invention contemplates a method of enhancing antimicrobialdefense, comprising administering an effective amount of a therapeuticcomposition to a host suspected of having a microbial infection, thecomposition comprising a product of the 5-lipoxygenase pathway. Inaddition, the present invention contemplates a method of enhancingantimicrobial defense, comprising administering an effective amount of atherapeutic composition to a host for prophylactic purposes, thecomposition comprising a product of the 5-lipoxygenase pathway. Suchprophylactic administration is most frequently performed with patientswho are at high risk for developing a microbial infection. Patients whoare at high risk include, but are not limited to, patients with the AIDSvirus and other patients who are immunocompromised.

In one embodiment, the microbial infection is bacterial pneumonia. Inparticular embodiments, the product of the 5-lipoxygenase pathwaycomprises a leukotriene. When the product of the 5-lipoxygenase pathwaycomprises a leukotriene, the leukotriene is leukotriene B₄ in certainembodiments and a cysteinyl leukotriene (e.g., leukotriene C₄,leukotriene D₄, and leukotriene E₄) in other embodiments. In stillfurther embodiments, the method of administering comprises pulmonaryadministration, and the pulmonary administration is by aerosolization ofthe therapeutic composition in other embodiments. Moreover, certainembodiments further involve the co-administration of an antibiotic tothe host. The host is an animal in some embodiments, and a human inothers.

Furthermore, the present invention contemplates a method of treating abacterial infection, comprising administering an effective amount of atherapeutic composition to a host having a bacterial infection, thetherapeutic composition comprising a leukotriene. In particularembodiments, the bacterial infection is bacterial pneumonia. Theleukotriene is leukotriene B₄ in certain embodiments and a cysteinylleukotriene like leukotriene C₄, leukotriene D₄, and leukotriene E₄ inother embodiments. In still further embodiments, the method ofadministering comprises pulmonary administration, and the pulmonaryadministration is by aerosolization of the therapeutic composition inother embodiments. Moreover, certain embodiments further involve theco-administration of an antibiotic to the host. The host is an animal insome embodiments, and a human in others.

Finally, the present invention contemplates a solution for the treatmentof a microbial infection, the solution comprising a sterile liquidvehicle and a leukotriene dissolved in the sterile liquid vehicle. Inparticular embodiments, the leukotriene is leukotriene B₄. In stillfurther embodiments, the leukotriene is a cysteinyl leukotriene; whenthe leukotriene is a cysteinyl leukotriene, it is leukotriene C₄,leukotriene D₄, or leukotriene E₄ in particular embodiments. Finally,the solution is aerosolized in still additional embodiments.

DEFINITIONS

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The phrase "product of the 5-lipoxygenase pathway" refers to thosecompounds that result from the enzymatic conversion of arachidonic acidby 5-lipoxygenase. Products of the 5-lipoxygenase pathway include5-hydroperoxyeicosatetraenoic acid 5-HPETE! and LTA₄, as well ascompounds derived therefrom. The products encompass 5-HETE, which isproduced from 5-HPETE. The products also include compounds formed fromthe conversion of LTA₄, such as LTB₄, LTC₄, LTE₄, and LTF₄. Moreover,the products are meant to encompass derivatives (i.e., compoundsproduced by structural modification) of compounds produced in thearachidonic acid cascade. The present invention is not limited by thenature of the structural modification; modifications include, but arenot limited to, the formation of a double bond between two carbon atomsand the addition of functional groups like hydroxyl and carboxymoieties. Further modifications contemplated by the present inventioninclude the substitution of different amino acids for those normallypresent (e.g., the replacement of the glycine residue on LTD₄ withanother amino acid) or the attachment of additional amino acids. Thefollowing table (Table 1) lists various commercially available products(Cayman) of the 5-lipoxygenase pathway. Of course, the present inventionis not limited to those compounds set forth in Table 1.

                  TABLE 1    ______________________________________    Parent Compound                 Derivative Compounds    ______________________________________    Leukotriene A.sub.4                 Leukotriene A.sub.4 methyl ester    Leukotriene B.sub.3    Leukotriene B.sub.4                 Leukotriene B.sub.4 -d.sub.4                 Leukotriene B.sub.4 dimethyl amide                 6-trans Leukotriene B.sub.4                 6-trans-12-epi Leukotriene B.sub.4                 12-epi Leukotriene B.sub.4                 18-carboxy dinor Leukotriene B.sub.4                 20-carboxy Leukotriene B.sub.4                 20-hydroxy Leukotriene B.sub.4    Leukotriene B.sub.5    Leukotriene C.sub.4    Leukotriene C.sub.5    Leukotriene D.sub.4    Leukotriene D.sub.5    Leukotriene E.sub.4                 N-acetyl Leukotriene E.sub.4                 16-carboxy-Δ.sup.13 -tetranor Leukotriene E.sub.4                 N-acetyl-16-carboxy-Δ.sup.13 -tetranor LTE.sub.4                 fluoro Leukotriene E.sub.4    Leukotriene E.sub.5    Leukotriene F.sub.4    Leukotriene Mixtures                 Peptido-Leukotriene Mixtures                 Leukotriene A.sub.4 Metabolite Mixture                 Leukotriene E.sub.4 Metabolite Mixture    ______________________________________

The term "leukotriene" is herein defined functionally as those compoundscausing enhancement of antimicrobial defense. The term "microbial"includes, but is not limited to, bacteria, viruses, parasites, andfungi.

The term "cysteinyl leukotriene" refers to those leukotrienes thatpossess the cysteine residue characteristic of leukotrienes C₄, D₄, andE₄.

The term "eicosanoid" refers to compounds derived from 20-carbonessential fatty acids that contain three, four, or five double bonds:8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid),5,8,11,14-eicosatetraenoic acid (arachidonic acid), and5,8,11,14,17-eicosapentaenoic acid. The families of leukotrienes andprostaglandins are examples of eicosanoids.

The term "effective amount" refers to that amount of a 5-lipoxygenaseproduct that is required to successfully perform a particular function.Generally speaking, the effective amount of a 5-lipoxygenase productwill be that amount that enhances or improves (to any degree) theability of the body to eradicate a microbial infection, especially abacterial infection. The effective amount may depend on a number offactors, including the type of microbe involved, the severity of theinfection, the immune status of the individual, and the weight of theindividual. By way of example, leukotriene LTD₄ may be administered in atherapeutic composition containing between 0.1 μg and 10 μg.

The term "therapeutic composition" refers to a composition thatcomprises a product of the 5-lipoxygenase pathway (e.g., LTB₄ and LTC₄)in a pharmaceutically acceptable form. The characteristics of the formwill depend on a number of factors, including the mode ofadministration. For example, a composition for aerosolized pulmonaryadministration must be formulated such that the product ispharmacologically active following delivery to the lungs. Thetherapeutic composition may contain diluents, adjuvants and excipients,among other things. In a preferred embodiment, the product of the5-lipoxygenase pathway is dissolved in a sterile liquid vehicle. Theterm "sterile liquid vehicle" refers to those liquids that are suitablefor administration to a host (e.g., pulmonary or parenteraladministration) and allow dissolution of the product of the5-lipoxygenase pathway. Examples of sterile liquid vehicles include, butare not limited to, sterile normal saline and dilute concentrations ofethanol.

The term "host" refers to humans and animals.

The terms "enhancing microbial defense" and "enhancing bacterialdefense" refer broadly to the improved ability of a subject's immunesystem to respond to and eradicate a microbial infection (e.g., abacterial, parasitic, viral, and fungal infection) and specifically abacterial infection, respectively. The terms include, for example,augmentation of the subject's endogenous defense mechanisms. Thepresence of enhancement of antimicrobial/antibacterial defense isdetermined by subjecting a compound to the screening procedure describedin Table 3 below.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic depicting the pathway of leukotriene synthesisand the structures of the main products of the 5-lipoxygenase metabolicpathway.

FIG. 1B is a schematic overview depicting the pathway of leukotrienesynthesis and the actions of leukotrienes relevant to antimicrobialdefense.

FIGS. 2A and B depict RP-HPLC profiles of radioactive eicosanoidsreleased by prelabeled alveolar macrophages obtained from wild type mice(FIG. 2A) and 5-LO knockout mice (FIG. 2B).

FIG. 3 graphically depicts the effect of Klebsiella pneumoniae challengeon survival in 5-LO knockout mice and wild type mice.

FIG. 4 graphically depicts the clearance of K. pneumoniae from lung andplasma two days after challenge in 5-LO knockout and wild type mice.

FIG. 5 graphically depicts phagocytic and bactericidal activities inalveolar macrophages from 5-LO knockout and wild type mice.

FIG. 6 graphically depicts the effect of exogenous LTB₄ on bacterialphagocytic activity in alveolar macrophages from 5-LO knockout mice.

FIG. 7 graphically depicts lung homogenate levels of LTB₄ and LTC₄ inwild type mice two days after challenge with either K. pneumoniae orsaline.

FIG. 8 graphically depicts the effect of K. pneumoniae challenge onlavage neutrophilia in 5-LO knockout and wild type mice.

FIG. 9 graphically depicts the effect of exogenous 5-LO metabolites onbacterial phagocytic activity in normal rat alveolar macrophages.

FIG. 10 graphically depicts the effect of intratracheal administrationof LTB₄ on defective bacterial clearance of the lung in 5-LO knockoutmice.

DESCRIPTION OF THE INVENTION

The present invention relates generally to the administration ofcompounds to enhance microbial defense, and more particularly to theadministration of products of the 5-lipoxygenase metabolic pathway toenhance bacterial defense and to treat and prevent bacterial pneumonia.To facilitate an understanding of the present invention, the descriptionthat follows is divided into the following sections: I) Synthesis,Actions, and Pharmacologic Modulation of Leukotrienes; II) Leukotrienesand Antimicrobial Host Defense; III) 5-LO Activation in AlveolarMacrophages and Neutrophils; IV) Role of Leukotrienes in the In VivoHost Response; and V) Composition and Administration of Compounds.

I. Synthesis, Actions, and Pharmacologic Modulation of Leukotrienes

Leukotrienes are oxygenated derivatives of arachidonic acid synthesizedmainly by bone marrow-derived cells in response to a variety of solubleor particulate stimuli. E. Goetzl et al., FASEB J 9:1051-1058 (1995)!.Arachidonic acid is initially hydrolyzed from membrane phospholipids, inpart by the actions of cytosolic phospholipase A₂ (cPLA₂). The next twosteps in leukotriene synthesis (the sequential conversion of arachidonicacid first to 5-hydroperoxyeicosatetraenoic acid 5-HPETE! and then toLTA₄) are catalyzed by the enzyme 5-lipoxygenase (5-LO). This enzymeresides within the cytosol and/or the nucleus of resting cells. Thoughan understanding of its mechanism of action is not required in order topractice the present invention, upon agonist stimulation, it is believedthat 5-LO translocates in a Ca²⁺ -dependent manner to the nuclearenvelope see, e.g, J. Woods et al., J. Clin. Invest. 95:2035-2040(1995)!; here it is thought to gain access to free arachidonic acid,hydrolyzed from nuclear envelope phospholipids and presented by theintegral nuclear envelope arachidonic acid-binding protein, 5-LOactivating protein (FLAP). 5-HPETE can be converted to the stableproduct, 5-HETE. The LTA₄ can be enzymatically converted to LTB₄ (byLTA₄ hydrolase) or to LTC₄ (by LTC₄ synthase). In turn, LTC₄ can beenzymatically converted to LTD₄ (with concomitant increase inbioactivity) and then to LTE₄ ; LTE₄ may be subsequently modified toform LTF₄. FIG. 1A is a schematic depicting the pathway of leukotrienesynthesis and the structures of the main products of the 5-lipoxygenasemetabolic pathway; importantly, practice of the present invention doesnot depend on the accuracy of the model depicted in FIG. 1A.

Cellular leukotriene synthetic capacity can be enhanced by exposure to anumber of biologically active substances, such as granulocyte-macrophagecolony-stimulating factor, interferon-γ, and transforming growthfactor-β. As described in further detail below, alveolar macrophageshave a greater capacity for 5-LO metabolism than do blood monocytes orother tissue macrophages see, e.g., M. Peters-Golden et al., J. Immunol.144:263-270 (1990)!, and they produce both LTB₄ and LTC₄. Neutrophils,by contrast, produce only LTB₄. Alveolar macrophages and neutrophilsboth produce 5-HETE.

Though an understanding of their mechanism of action is not required inorder to practice the present invention, the principle bioactiveleukotrienes, LTB₄ and the cysteinyl or sulfidopeptide leukotrienes(leukotrienes C₄, D₄, and E₄), are thought to act by interacting withspecific surface receptors on target cells. LTB₄ is a potent neutrophilchemotaxin in vitro, accounting for the majority of chemotactic activityelaborated acutely by stimulated human alveolar macrophages in culture.T. Martin et al., J. Clin. Invest. 80:11 14-1124 (1989)!. In addition,in vivo bronchoscopic instillation of LTB₄ into the human lung resultedin neutrophil influx. T. Martin et al., J. Clin. Invest. 80:1009-1019(1989)!.

While the practice of the present invention does not depend on a preciseunderstanding of the effects of the products of the 5-LO pathway, LTB₄is thought to enhance numerous leukocyte functions, includingphagocytosis T. Demitsu et al., Int. J. Immunopharmac. 11:801-808(1989)!, upregulation of cell surface CR3 molecules P. Marder et al.,Biochem. Pharmacol. 49:1683-1690 (1995)!, the secretion of O₂ - andlysosomal hydrolases, mobilization of intracellular Ca²⁺ stores C.Serhan et al., Biochem. Biophys. Res. Commun. 107:1006-1012 (1982)!,phospholipase-dependent arachidonic acid release J. Wijkander et al., J.Biol. Chem. 270:26543-26549 (1995)!, activation of PKC J. O'Flaherty etal., J. Immunol. 144:1909-1913 (1990)!, the synthesis of interleukin(IL)-8 R. Strieter et al., Am. J. Pathol. 141:397-407 (1992)!, andactivation of natural killer cell activity R. Bray and Z. Brahmi Z, J.Immunol. 136:1783-1790 (1986)!. It is believed that 5-HETE shares manyof these same actions, but with less potency. The cysteinyl leukotrienespossess the bioactivity previously identified as slow reactingsubstance. Their most potent actions include constriction of bronchialand vascular smooth muscle and increasing microvascular permeability.LTD₄ has also been reported to increase macrophage FcR expression invitro J. Rhodes et al., Eur. J. Immunol. 15:222-227 (1985)! and actinpolymerization M. Peppelenbosch et al., Cell 74:565-575 (1993)!.

Though an understanding of the molecular mechanisms of bacterialingestion and killing by phagocytes is not required in order to practicethe present invention, the phagocyte surface receptors which are mostcritical for efficient opsonic phagocytosis are those which recognizethe Fc portion of IgG (FcRII and FcRIII) and the C3bi fragment ofcomplement (the integrin CR3, also known as Mac-1 and CD11b/CD18). CR3also mediates nonopsonic ingestion of K. pneumoniae. One consequence ofreceptor ligation is the release and metabolism of arachidonic acid.Because CR3 and FcR mediate attachment of K. pneumoniae to phagocytes,their surface expression are relevant targets for modulation byleukotrienes.

FIG. 1B is a schematic depicting the pathway of leukotriene synthesisand the actions of leukotrienes relevant to antimicrobial defense.Bacteria, such as K pneumoniae, attach to phagocytic cells such asalveolar macrophages and neutrophils and are phagocytosed. It isbelieved that this triggers an increase in intracellular Ca²⁺, which inturn results in translocation of cPLA₂ and 5-LO to the nuclear envelope.As previously indicated, arachidonic acid is hydrolyzed fromphospholipids and metabolized by 5-LO, interacting with FLAP, to LTA₄.LTA₄ is further converted to leukotrienes B₄ and C₄. These may affecttarget cells, via interactions with receptors, in either autocrine orparacrine fashion. As a result, chemotaxis, bacterial phagocytosis, andbacterial killing are promoted.

Interruption of the synthesis or actions of leukotrienes has been aprime therapeutic target of the pharmaceutical industry. Potent andspecific compounds are now available which inhibit leukotriene synthesisby directly inhibiting either 5-LO or FLAP; both classes of agentsinhibit synthesis of all 5-LO products. In addition, compounds whichspecifically antagonize the LTB₄ and cysteinyl leukotriene receptors arealso available; unlike the former class, these agents offer thecapability to block the actions of individual leukotrienesindependently. Although preclinical studies suggest a variety ofpotential disease targets, asthma has received the most attention inclinical studies.

II. Leukotrienes and Antimicrobial Host Defense

It is generally assumed that inflammatory cascades have evolved for thepurpose of host defense against microbial invasion. Yet little is knownabout the possible importance of endogenous leukotrienes in mediatingthe host response to infection. The increasing incidence ofimmunosuppression and the emergence of antibiotic-resistant microbesunderscore the importance of understanding the innate host defensemechanisms.

The sterility of the pulmonary alveolar surface is under constantassault by inhaled and aspirated microbes. Effective clearance of thesepathogens depends largely on innate immune responses involving microbialphagocytosis and killing. Prior to the work of the present inventors,researchers have largely ignored the products of the 5-LO pathway aspotentially representing a class of inflammatory mediators inantimicrobial defense.

The present inventors have found that exogenously administered productsof the 5-LO pathway in general, and leukotrienes in particular, areassociated with a number of possible advantages as adjunctive agents inthe treatment of pneumonia. Specifically, the inventors have determinedthat these products exhibit a rapid onset of action and believe thatthese products do not elicit immunologic responses in the recipient.Moreover, such products represent a relatively inexpensive therapy thatcan be used independent of antibiotics or as adjunct therapy toantibiotics in the treatment of pneumonia. Particular patientpopulations (e.g., patients with AIDS, diabetes, smokers, neonates, andpatients suffering from alcoholism and malnutrition) with severepneumonia would benefit from augmenting endogenous host defensemechanisms through the rational administration of, for example,leukotrienes to the lungs.

Though a precise understanding of the effects of leukotrienes onantimicrobial host defense is not required to practice the presentinvention, it is believed that certain general effects occur. First,endogenous leukotrienes must be present at sites of infection in orderto participate in antimicrobial defense, and increased (relative tocontrols) levels of LTB₄ have been measured in bronchoalveolar lavagefluid (BALF) and lung tissue of Pseudomonas aeruginosa-infected rats A.Buret et al., Infect. Immun. 61:671-679 (1993)! as well asbronchoalveolar lavage fluid of patients with bacterial pneumonia H.Hopkins et al., Chest 95:1021-1027 (1989)!. As described in furtherdetail below, the present inventors have also measured high levels ofboth LTB₄ and LTC₄ in lung homogenates of mice with Klebsiellapneumonia. Klebsiella pneumoniae is the classic cause of Gram-negativepneumonia and has been reported to account for 18-64% ofcommunity-acquired and 30% of nosocomial Gram-negative pneumonias. L.Crane and A. Lerner, In: Respiratory Infections: Diagnosis andManagement (J. Pennington, ed.) (Raven Press, New York), pp. 227-250(1983)!.

Second, exogenously added leukotrienes enhance microbial phagocytosisand/or killing. As described above, the addition of LTB₄ promotesneutrophil chemotaxis as well as phagocytosis of particles, signaltransduction, and secretion of oxidants and lysosomal enzymes--all ofwhich would be expected to facilitate bacterial clearance. Indeed, LTB₄enhanced the in vitro phagocytosis and killing of P. aeruginosa andSalmonella typhimurium by peritoneal macrophages T. Demitsu et al., Int.J. Immunopharmac. 11:801-808 (1989)!, and the in vitro killing ofSchistosoma mansoni by neutrophils R. Moqbel et al., Clin. Exp. Immunol.52:519-527 (1983)!; intraperitoneal injection of LTB₄ also enhanced thein vivo clearance of S. typhimurium administered by the same route T.Demitsu et al., Int. J. Immunopharmac. 11:801-808 (1989)!. However,prior to the present invention, it is believed that the intrapulmonaryadministration of leukotrienes and other products of the 5-LO pathwayhas not previously been reported for therapeutic use.

Third, reduction of endogenous leukotriene synthesis increasessusceptibility to infection. Phagocytosis, degranulation, and nitricoxide production have been reported to be inhibited by relativelyspecific inhibitors of 5-LO, indicating a permissive role for endogenousleukotrienes in these functions. Interestingly, a number of situationscharacterized by predisposition to pulmonary infections are associatedwith a reduced in vitro capacity of alveolar macrophages to synthesizeleukotrienes; these include both human conditions (HIV infection andsmoking) as well as animal models (proteincalorie malnutrition, vitaminD deficiency, and the neonatal period). A similar association has beennoted for peripheral blood leukocytes from patients with diabetesmellitus. This raises the possibility that a defect in 5-LO metabolismcould underlie the multiple defects in leukocyte function which havebeen demonstrated in poorly controlled diabetics. Clinical use ofanti-leukotriene agents in asthma has not been associated with anincrease in respiratory infections. However, these studies havegenerally been short-term (i e., several weeks or months), and youngotherwise healthy asthmatics are not a patient population which would beexpected to be predisposed to such infections.

III. 5-LO Activation in Alveolar Macrophages and Neutrophils

Alveolar macrophages have been demonstrated to have a greater capacityfor leukotriene synthesis than peripheral blood monocytes or othertissue macrophages. This is the situation in response to both soluble(ionophore A23187) and particulate (zymosan) agonists, and for cellsfrom humans M. Balter et al., J. Immunol. 142:602-608 (1989)! as well asrats M. Peters-Golden et al., J. Immunol. 144:263-270 (1990)! (data notshown). Moreover, as described further in the Experimental section, theprofile of eicosanoids released by stimulated murine alveolarmacrophages is likewise comprised largely of 5-LO metabolites (see FIG.2A).

The present inventors have also demonstrated that neutrophils recruitedto sites of inflammation exhibit increased leukotriene syntheticcapacity and a shift in intracellular 5-LO distribution. Indeed, thepresent inventors have compared leukotriene synthetic capacity andintracellular distribution of 5-LO in rat neutrophils isolated fromperipheral blood or from peritoneal lavage fluid 4 hours after glycogeninstillation. Elicited neutrophils exhibited a 5-fold greater maximalcapacity for LTB₄ synthesis in response to A23187 than did bloodneutrophils studied in parallel (data not shown). In addition, the twocell populations exhibited strikingly different intracellulardistributions of 5-LO in the resting state. As previously demonstratedfor human blood neutrophils T.G. Brock et al., J. Biol. Chem.269:22059-22066 (1994)!, the resting rat blood neutrophils contained5-LO exclusively in the cytosol. By contrast, the resting elicitedneutrophils contained a substantial proportion of their 5-LO within thenucleus; upon subsequent ionophore activation, both blood and elicitedneutrophil populations showed 5-LO translocation to the nuclear envelope(data now shown).

In addition to the findings, described above, with neutrophils recruitedto the peritoneum, the present inventors have also observed apredominant intranuclear localization of 5-LO in neutrophils recruitedto the alveolar space, as evidenced in rats studied 2 dayspost-intratracheal administration of bleomycin. This can be demonstratedboth by immunofluorescence microscopic analysis of lavage cells and byimmunohistochemical staining of lung sections (data not shown). Intotal, these results suggest that, in the process of recruitment fromthe bloodstream to diverse anatomic sites of inflammation, neutrophilsi) import cytosolic 5-LO into the cell nucleus and ii) upregulate theirmaximal capacity for leukotriene generation. In both of these respects,recruited neutrophils resemble alveolar macrophages.

Importantly, it is known that there is reduced leukotriene syntheticcapacity in alveolar macrophages from humans or animals predisposed topulmonary infections. The present inventors have examined the 5-LOmetabolic capacity of alveolar macrophages isolated from various humanor animal conditions known to be associated with increasedsusceptibility to pulmonary infections. These conditions includedcontrolled studies with human subjects who smoke M. Balter et al., J.Lab. Clin. Med. 114:662-673 (1989)!, human subjects infected with thehuman immunodeficiency virus (CD4 count less than 200) M. Coffey et al.,J. Immunol. 157:393-399 (1996)!, vitamin D-deficient rats M. J. Coffeyet al., Prostaglandins 48:313-329 (1994)!, newborn calves, andalcohol-fed mice. In all cases, the subjects had no evidence ofbacterial lung infections at the time of study. In each of thesecircumstances, the in vitro capacity of alveolar macrophages forleukotriene synthesis was reduced by 60-90% as compared to the controllevels. These findings indicate that the administration of exogenousleukotrienes should enhance the host defense mechanism in patientssusceptible to lower respiratory tract infections.

IV. Role of Leukotrienes in the In Vivo Host Response

The development of leukotriene-deficient mice by targeted disruption ofthe 5-LO gene represents an important tool to evaluate the role ofendogenously generated leukotrienes. J. Goulet et al., Proc. Natl. Acad.Sci. USA 91:12852-12856 (1994); X. Chen et al., Nature 372:179-182(1994)!. These knockout mice have been found to have a reduced abilityto recruit neutrophils in most models of inflammation. The presentinventors tested commercially available 5-LO knockout mice to furthershow that impaired endogenous leukotriene synthetic capacity is causallyrelated to impaired antimicrobial defense of the lung.

The present inventors used Klebsiella pneumoniae as a causative pathogento induce pneumonia for several reasons. First, as previously discussed,it is of great clinical relevance in pneumonia. Second, it causes abrisk inflammatory response in mice. A. McColm et al., J. Antimicrob.Chemother. 18:599-608 (1986)!. Third, the murine K. pneumoniae model hasbeen extensively characterized by one of the co-inventors. In theexperiments described below, intratracheal (i.t.) injection was utilizedrather than aerosolization because it more closely resembles the bolusof organisms which reaches the distal lung via microaspiration.Following intratracheal challenge of CD-1 mice with 10³ CFU of K.pneumonia, neutrophil influx peaks at 48 hours and most animals havedied by day 5. In addition, lung homogenate levels of various cytokinesincrease and also peak at 48 hours; these include tumor necrosis factor(TNF), macrophage inflammatory protein-2 (MIP-2), macrophageinflammatory protein-1α (MIP-1α), IL-12, and IL-10.

In order to apply this model of pneumonia to 5-LO knockout mice, thepresent inventors first identified an inoculum of organisms which wasappropriate for the wild type background strain, 129/SvEv. This strainof mice proved to be even more susceptible to Klebsiella pneumonia thanthe CD-1 strain. Specifically, previous studies determined thatapproximately 50% mortality occurred in the wild type animals with abacterial inoculum of only 50 CFU, indicating that 129/SvEv mice weresubstantially more susceptible to Klebsiella pneumonia than the CD-1strain. M. Greenberger et al., J. Immunol. 155:722 (1995)!. Therequirement for a low bacterial inoculum makes this a relevantexperimental model for Gram-negative pneumonia in humans, which isgenerally believed to result from microaspiration of oropharyngealcontents containing relatively small numbers of organisms.

Though the present invention utilizes a murine K. pneumonia model, thepresent invention is not limited to augmenting the treatment ofinfections caused by that organism. Indeed, the present inventioncontemplates the administration of products of the 5-LO metabolicpathway, particularly LTB₄ and LTC₄, independently and as an adjunct(e.g., with antibiotics) to the treatment of pneumonia and otherrespiratory tract infections caused by a panoply of organisms. Table 2lists some of the most common bacterial pathogens that causecommunity-acquired and hospital-acquired pneumonia. It is contemplatedthat patients with infections caused by these organisms will benefitfrom administration of the products of the 5-LO metabolic pathway.

                  TABLE 2    ______________________________________    Type of Pneumonia                Type of Pathogen    ______________________________________    Community Acquired                most frequent:                Streptococcus pneumoniae                Haemophilus influenzae                Mycoplasma pneumoniae                less frequent:                Staphylococcus aureus                Legionella sp.                Gram-negative bacilli (alcoholics)                aspiration:                mouth anaerobes (e.g., Peptococci spp.)    Hospital Acquired                most frequent:                Enterobacteriaceae (e.g., Klebsiella spp., E. coli)                Pseudomonas aeruginosa                Staphylococcus aureus                aspiration:                mouth anaerobes    ______________________________________

The present invention is not limited to augmentation of the treatment ofpneumonia. Indeed, the present invention contemplates the administrationof products of the 5-LO metabolic pathway as therapy in the treatment ofother infections that have pulmonary manifestations. Moreover, asalluded to above, the present invention contemplates the administrationof the products for the treatment and prophylaxis of a broad range ofmicrobial infections besides bacterial infections, including infectionscaused by parasites R. Moqbel et al., Clin. Exp. Immunol. 52:519-527(1983)!, viruses, and fungi. Furthermore, the present inventioncontemplates augmentation of the treatment of systemic infections; itshould be pointed out that systemic administration should be performedcautiously, as the leukotrienes are known to cause hypotension.

Moreover, while the present invention contemplates in vivo pulmonaryadministration of leukotrienes and other 5-LO products to augmentdefense against bacteria in leukotriene-deficient hosts, the presentinvention also contemplates in vivo administration to patients who arenot leukotriene-deficient; indeed, such use is supported by the factthat in vitro incubation with exogenous leukotrienes augmentsphagocytosis and killing by normal macrophages.

Of note, the future use of anti-leukotriene drugs in humans is likely tomimic the leukotriene deficiency observed with 5-LO gene disruption inmice. In certain individuals who are on other immunosuppressive agentsor who have increased numbers of bacteria in their lower respiratorytracts, the use of such drugs may compromise pulmonary antimicrobialhost defense. As a result, these individuals may also benefit fromadministration of products of the 5-LO pathway contemplated for use withthe present invention; of course, particular dosing schedules andregimens may be warranted when these agents are used concomitantly withpatients taking anti-leukotriene drugs.

As previously alluded to, the present invention contemplates the use ofdiverse products of the 5-lipoxygenase metabolic pathway in order toenhance bacterial defense. The comprehensive screening procedure setforth in Table 3 can be used to evaluate those products (such as thosecompounds previously presented in Table 1), as well as derivatives oranalogues of such products, that may be effective. Leukotrienes B₄ andC₄ are particularly effective at enhancing bacterial defense, and thisscreen is especially appropriate for compounds related to thoseleukotrienes. Reference to a particular example is given with eachdetermination; the indicated examples provide a detailed description ofhow the determination is to be carried out.

                                      TABLE 3    __________________________________________________________________________    Step        Determination          Conclusion    __________________________________________________________________________    I   Measure in vitro the activity of compounds on                               Proceed to Step II with those        alveolar macrophage phagocytic and bactericidal                               compounds that increase        activities (see, e.g., Example 3).                               phagocytic and bactericidal                               activities.    II  Determine in vivo the effect of compounds on                               Proceed To Step III with those        bacterial clearance after 48 hours by measuring CFU                               compounds that increase        in lung homogenate (see, e.g., Example 2).                               clearance.    III Determine in vivo the effect of compounds via                               Proceed to Step IV with those        different routes of administration and administered                               compounds that exhibit efficacy        different time points post-bacterial challenge (see,                               following the administration via at        e.g., Example 10).     lease one route.    IV  Verify the findings of Step III by examining animal                               Consider clinical trials.        survival.    __________________________________________________________________________

As illustrated by this outline of the sequence of experimentalprocedures and the description of the procedures themselves, thoughtfulconsideration allows any compound (e.g., "Compound X") to be evaluatedfor use with the present invention. Indeed, as described in detail inthe Experimental section, these screening procedures have been employedin the experiments performed with LTB₄.

V. Composition And Administration Of Compounds

The present invention contemplates using therapeutic compositions ofproducts of the 5-LO metabolic pathway that are indicated as beingefficacious based on application of the screen described above. It isnot intended that the present invention be limited by the particularnature of the therapeutic preparation. For example, such compositionscan be provided together with physiologically tolerable liquid (e.g,saline), gel or carriers or vehicles, diluents, adjuvants andexcipients, such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like, and combinations thereof. These compositions typicallycontain 1%-95% of active ingredient, preferably 2%-70%. In addition, ifdesired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing or pHbuffering agents or preservatives. Generally speaking, the nature of thecomposition will depend on the method of administration.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts and the organism involved.

A preferred mode of administration comprises administration to the lung.Patients who are sick enough to require mechanical ventilation canreceive treatment with pharmacologic agents administered via theendotracheal tube which is connected to the ventilator. Alternatively,intrapulmonary delivery of pharmacologic agents to patients notrequiring mechanical ventilation can be accomplished via aerosolization.Alternatively, the agent may be administered to the lung through abronchoscope. Of course, the therapeutic agents may be investigated fortheir efficacy via other routes of administration, including parenteraladministration. However, when the site of infection is the lung,targeting drug delivery thereto is likely to minimize side effects andsystemic consequences.

In addition, the compounds contemplated by the present invention possessattributes as therapeutic agents over other agents like polypeptides.For example, the products of the 5-LO metabolic pathway contemplated bythe present invention have a rapid onset of action (generally within 1hour) and short duration of action (generally less than 12 hours); theseattributes permit a substantial degree of control over biologicaleffects. In addition, their short duration of action reduces thepossibility that administration of leukotrienes and related agents mightadversely stimulate an over-exuberant inflammatory response. Moreover,commercially-available leukotriene receptor antagonists (e.g., thecysteinyl antagonist Accolate® (zafirlukast) Zeneca) can be administeredto further prevent such an inflammatory reaction from occurring.

As previously alluded to, the products of the 5-LO metabolic pathwaycontemplated by the present invention are associated with additionalattributes. For example, the lipid products do not elicit immunologicreactions like polypeptide agents do. Furthermore, the compounds of thepresent invention are relatively inexpensive, making them ideal as anadjunct to infection treatment.

The compounds contemplated by the present invention provide a means forenhancing pulmonary defense capabilities. They are especiallyefficacious in the treatment and prevention of bacterial pneumonia inthose patients who are predisposed to that condition. Of course, thepresent invention contemplates the use of the compounds in the treatmentand prevention of other infections and ailments, alone or in combinationwith, for example, other products of the 5-LO pathway or antimicrobialagents.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (Molar); mM (millimolar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); L (liters); mL (milliliters); μL (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); min.(minutes); s and sec. (seconds); OD (outside diameter); °C. (degreesCentigrade); v/v (volume/volume); AM (alveolar macrophage); BAL(bronchoalveolar lavage); BALF (bronchoalveolar lavage fluid); cPLA₂(cytosolic phospholipase A₂); CFU (colony-forming unit); 5-LO(5-lipoxygenase); FLAP (5-LO activating protein); AA (arachidonic acid);LT (leukotriene); LTB₄ (leukotriene B₄); LTC₄ (leukotriene C₄); LTB₄ R(LTB₄ receptor); cys-LTR (cysteinyl leukotriene receptor); CR3(complement receptor 3); FcR (receptor for Fc portion of Ig); MPO(myeloperoxidase); PKC (protein kinase C); MAPK (Mitogen ActivatedProtein Kinase); O₂ -(superoxide); NO (nitric oxide); PM (peritonealmacrophages); PMN (polymorphonuclear leukocytes); KO (knockout); WT(wild type); TNF (tumor necrosis factor); JE (the murine homologue ofmonocyte chemotactic peptide-1); IL (interleukin); HBSS (Hank's BalancedSalt Solution); RP-HPLC (reversed-phase high pressure liquidchromatography); SE (standard error); SEM (standard error of the mean);Abacus (Abacus Concepts, Inc., Berkeley, Calif.); Abbott (AbbottLaboratories, North Chicago, Ill.); ATCC (American Type CultureCollection; Rockville, Md.); Baxter (McGaw Park, Ill.); Biogenics (Napa,Calif.); Cayman (Cayman Chemical; Ann Arbor, Mich.); Coulter (CoulterCorp., Miami, Fla.); Difco (Detroit, Mich.); Fisher Scientific,Pittsburg, Pa.); Gibco (Gibco BRL; Gaithersburg, Md.); Jackson (TheJackson Laboratory; Bar Harbor, Me.); Merck (Rahway, N.J.); MolecularProbes (Eugene, Oreg.); Nunc (Naperville, Ill.); PharMingen (San Diego,Calif.); Pierce (Rockford, Ill.); Pfizer (Pfizer Inc., New York, N.Y.);Vector (Vector Laboratories, Burlingame, Calif.); and Waters (WatersCorp., Milford, Mass.); Zeneca (Zeneca Pharmaceuticals, Wilmington,Del.).

The following General Methods were used in the examples that followunless otherwise indicated.

Animals

Mice with the targeted disruption of their 5-LO gene (ALOX 5, designatedKO) and their wild type strain controls (129/SvEv, designated WT) wereobtained from The Jackson Laboratory.

K. pneumoniae Inoculation

K. pneumoniae strain 43816, serotype 2 obtained from the ATCC (AssessionNo. 29939) was grown in tryptic soy broth (Difco) for 18 hours at 37° C.The preparation and intratracheal administration of K. pneumoniae werecarried out as described by M. Schneemann et al. J. Infect. Dis.167:1358-1363 (1993)!. Bacterial concentration was determined bymeasuring absorbance at 600 nm and referencing to a standard curve ofabsorbances vs. known standard CFUs. Bacteria were then pelleted bycentrifugation for 30 min at 10,000 rpm, washed×2 in saline, andresuspended at the desired concentration in saline.

After appropriate dilution of bacteria in endotoxin-free saline, animalswere anesthetized with sodium pentobarbital (approximately 0.2 mLdiluted 1:7 in saline intraperitoneally) and the trachea was exposed viaa small midline incision. A 30 μL inoculum containing 50 CFU K.pneumoniae or saline was administered via a sterile 26-gauge needle andthe skin was closed with a surgical staple. For preparation of K.pneumoniae-specific serum, wild type mice are similarly anesthetized andinoculated intratracheally (with 25 CFU bacteria); animals are bledorbitally 2 weeks later, and serum obtained.

Determination of Plasma and Lung CFU

Plasma and lung CFU were determined as described by M. Schneemann et al.J. Infect. Dis. 167:1358-1363 (1993)!. Briefly, lungs homogenized in 3mL sterile saline and plasma collected at euthanasia were placed on ice,and serial 1:10 dilutions made. Ten μL of each dilution were plated onsoy base blood agar plates (Difco), incubated for 18 hours at 37° C.,and colonies were enumerated.

Preparation and Analyses of Lung Homogenates

At 30 and 48 hours post-inoculation, mice were anesthetized and bloodwas collected by orbital exsanguination. The mice were then euthanizedvia cervical dislocation and whole lungs were harvested for thedetermination of cytokine levels, myeloperoxidase activity (MPO), andleukotriene levels. For cytokine and leukotriene analyses, lungs werehomogenized in 2 mL of buffer containing 0.5% Triton X100, 150 mM NaCl,15 mM Tris-HCl, 1 mM CaCl₂, and 1 mM MgCl₂. Homogenates were thencentrifuged at 1500×g for 10 minutes and supernatants filtered through a1.2 μm syringe filter and immediately frozen at -20° C. TNF, macrophageinflammatory protein-1α, macrophage inflammatory protein-2, murine JE,and IL-12 were each quantified using a modification of a double ligandmethod as described by M. Schneemann et al. J. Infect. Dis.167:1358-1363 (1993)!. For determination of leukotriene levels in lunghomogenates, samples were extracted on C₁₈ Sep-Pak® cartridges® (Waters)to remove potentially cross-reactive materials, and evaporated todryness under N₂. J. Wilborn et al., J. Clin. Invest. 97:1827 (1996)!.An analogous procedure is used with bronchoalveolar lavage fluid.

Samples were resuspended in assay buffer and LTB₄ and LTC₄ levels weredetermined according to manufacturers instructions using enzymeimmunoassay kits obtained from Cayman Chemical. MPO activity, an indexof neutrophil influx, was quantified in lung homogenates as described byM. Greenberger et al. J. Immunol. 155:722 (1995)!. Briefly, lungs werehomogenized in 2 mL of buffer containing 50 mM potassium phosphate, pH6.0, with 5% hexadecyltrimethylammonium bromide and 5 mM EDTA. Thehomogenate was sonicated and centrifuged and the supernatant was mixed1:15 with assay buffer (86 mM monobasic sodium phosphate, 12 mM dibasicsodium phosphate, 0.0005% v/v! H₂ O₂, and 0.167 mg/mL o-dianisidinehydrochloride) and read at 490 nm (Beckman DU-64). MPO units werecalculated as the change in absorbance over time. Protein content ofhomogenates is determined using a microtiter plate modification (PierceBiochemical) of the Bradford method using bovine serum albumin as astandard.

Lung Lavage

The trachea was exposed through a 0.5 cm incision and intubated using a1.7 mm OD polyethylene catheter. Bronchoalveolar lavage was performed byinstilling 1 mL aliquots of phosphate-buffered saline containing 5 mMEDTA. Approximately 4 mL of lavage fluid were retrieved per mouse, andtotal cell numbers and differential cell counts were determined fromcytospins on each sample.

Alveolar Macrophage Culture and Functional Assays

For assays of bacterial phagocytosis and killing, alveolar macrophageswere purified from bronchoalveolar lavage cells by adherence for 1 hourin HBSS and studied in monolayer culture. Adherent cells werepreincubated with 5% K. pneumoniae-specific immune serum (as a source ofboth complement and specific opsonizing antibody) for 5 minutes at 37°C. prior to assays. Phagocytosis was studied by incubating 10⁵ alveolarmacrophages with 10⁶ K. pneumoniae in each well of an 8-well Labtek®plate (Nunc) for 1 hour at 37° C.; in some experiments, exogenous LTB₄(Cayman) was added concomitantly with bacteria. The supernatants wereaspirated and the cells were washed 3 times with HBSS. The slides werethen allowed to air dry, Diff-Quik® (Difco) staining was performed, and200 cells per well were counted to determine number of intracellular K.pneumoniae and percent of alveolar macrophages containing bacteria.Phagocytic index was calculated as the mean percentage of alveolarmacrophages containing bacteria multiplied by the mean number ofbacteria per alveolar macrophage.

The bactericidal activity was assayed by incubating for 1 hour at 37° C.the same numbers of alveolar macrophages and organisms as detailedabove, but in 35 mm tissue culture dishes. Supernatants were removed andcells were than washed with HBSS and lysed by adding 1 mL of coldsterile water, scraping with a rubber policeman, and incubating on icefor 10 minutes. One mL of 2×HBSS was added to each plate and lysateswere serially diluted on blood agar plates. Plates were incubated for 18hours at 37° C. and colony counts performed. Percent killing ofintracellular bacteria was calculated by the following formula:100--(number of bacterial CFU/mL alveolar macrophage lysate divided bythe total number of intracellular bacteria), where total intracellularK. pneumoniae is the product of the total number of alveolar macrophagesx the percentage of alveolar macrophages containing bacteria x the meannumber of bacteria per alveolar macrophage.

Neutrophil Culture and Functional Assays

To obtain peritoneal elicited neutrophils, mice are injectedintraperitoneally with 5% glycogen in PBS and peritoneal lavage isperformed 5 hours later. Approximately 3×10⁶ cells are obtained fromeach animal, approximately 85-90% of which are neutrophils. These cellsare likewise placed into culture for functional studies. Phagocytic andbactericidal assays are performed as described above.

Lung lavage from K. pneumoniae-challenged animals yields a mixture ofalveolar macrophages and neutrophils; they are found in a ratio ofapproximately 1:1 at 2 days post-inoculation, but the ratio is likely tovary over time. To determine constitutive secretion of leukotrienes bythese cells, mixed bronchoalveolar lavage cells are placed into culture(5×10⁵ cells/well) as described above for purified populations; theratio of alveolar macrophage:neutrophil in adherent monolayers isdetermined by direct Diff-Quik® staining of monolayers after the removalof medium. In all instances where leukotriene levels in culture mediumare quantitated, values are expressed per μg of cell protein. Culturemedium is medium 199 (Gibco).

In Vivo Administration of Anti-leukotriene Drugs and Leukotrienes

Doses of 5-LO inhibitor (A-79175; Abbott), LTB₄ receptor antagonist(CP-105,696; Pfizer), and cysteinyl leukotriene receptor antagonist(MK-571; Merck Research Laboratories) are suspended in methylcelluloseand administered once per day orally to unanesthetized mice using a 22gauge gavage needle.

Ethanolic stock solutions of LTB₄, LTC₄, and 5-HETE (Cayman Chemical)were diluted in saline and a 10 μL volume used for intratrachealinjection. For nebulization, a particle size <3 μm and a nose-onlyexposure chamber is utilized.

Immunohistochemical Staining for 5-LO

Lung sections as well as bronchoalveolar lavage cytospins are stainedfor 5-LO in order to identify the frequency and types of cellsexhibiting localization of enzyme to the nuclear envelope (an"activated" pattern). J. Wilborn et al., J. Clin. Invest. 97:1827-1836(1996)!. Briefly, specimens are fixed in 4% paraformaldehyde, embeddedin paraffin, and 3-μm-thick sections cut and mounted on Superfrost/PLUS®slides (Fisher Scientific). Paraffin is removed with Americlear®(Baxter) and tissue is rehydrated. To reduce nonspecific binding, tissueis incubated with Power Block® (Biogenics) followed by 25% normal goatserum.

Sections and cytospins are incubated at 4° C. for 24 hours with eitherrabbit anti-human 5-LO antiserum (Merck Frosst Canada) or nonimmunerabbit serum at 1:1000 in 25% normal goat serum in PBS. This antibodyalso recognizes the mouse and murine 5-LO. Goat anti-rabbit IgG (1:600)is then applied for 30 minutes and primary antibody is detected usingTrue-Blue® peroxidase substrate with Contrast Red® counterstain (bothfrom KPL Laboratories). The proportion of positively stained cellsexhibiting an activated pattern is determined from counts of 20 highpower fields. Cells staining positively for 5-LO (most of which areexpected to be either macrophages or neutrophils) are classified as tocell type on the basis of morphology. If necessary to distinguishalveolar macrophages and neutrophils, dual staining is undertaken. Celltype-specific staining is accomplished either with a second primary(e.g., anti-neutrophil antibody) or via histochemical staining (e.g, fornonspecific esterase or MPO). The second protein is detected by VectorRed® (Vector) to contrast with the True-Blue® stain for 5-LO.

Cell Surface Expression of CR3 and FcR Receptors

Expression of CR3 and FcR is quantitated in both alveolar macrophagesand neutrophils by staining with FITC-conjugated anti-mouse monoclonalantibodies with subsequent analysis by flow cytometry. L. Laichalk etal., FEMS Immunol. Med. Microbiol. 658:1-7 (1996)!. The FITC-conjugatedmonoclonal antibodies (PharMingen) include anti-CR3 IgG₁,anti-FcRII/FcRIII IgG₁, and an anti-IgG₁, isotype control. Experimentalincubations are carried out in suspension. Five×10⁵ cells are stainedwith 1 μg of monoclonal antibody for 30 minutes on ice, washed, fixed in2% paraformaldehyde in PBS, and stored at 4° C. in the dark untilanalyzed. Samples are analyzed on an EPICS C flow cytometer withaccompanying software (Coulter Corp.) available at the University ofMichigan Flow Cytometry Core Facility, examining at least 20,000 eventsper sample. After correction for staining by the control IgG, both thepercentage of positively stained cells and the mean fluorescenceintensity are determined.

Analysis of Actin Polymerization

Engulfment of attached particles or bacteria requires cytoskeletalrearrangement, including local actin polymerization. Polymerized actin(F-actin) is analyzed by staining with rhodamine-phalloidin (MolecularProbes) at a 1:300 dilution. Intracellular localization of F-actin isassessed by immunofluorescence microscopy. Cells on cover slips arefixed with formalin and permeabilized in acetone. T.G. Brock et al., J.Biol. Chem. 269:22059-22066 (1994)!. Following incubation withphalloidin for 1 hour, the cells are examined with a Nikon Labophot 2microscope equipped for epifluorescence. To quantify the total cellularcontent of F-actin, cells are permeabilized with 0.1% Triton X-100 andincubated with rhodamine-phalloidin and analyzed by flow cytometry. R.Crowell et al., Am. J. Respir. Cell Mol. Biol. 12:190-195 (1995)!.

Assessment of Phagosome-lysosome Fusion

Adherent cells from knockout or wild type mice are prelabeled byincubation for 15 minutes with 5 μg/mL acridine orange (MolecularProbes). Cells are washed, preincubated with specific immune serum, andthen incubated for up to 2 hours with K. pneumoniae alone or in thepresence of exogenous leukotrienes. Cells are examined byimmunofluorescence microscopy. Two hundred cells per condition arecounted, and the percentage of cells showing fusion as well as the totalnumber of fusion figures are recorded.

Assays for O₂ --, and β-glucuronidase

Superoxide production by 0.5-1.0×10⁶ adherent cells incubated with0.5-1.0×10⁷ K. pneumoniae or 100 nM phorbol myristate acetate isassessed from the superoxide dismutase-inhibitable reduction offerricytochrome C. L. Laichalk et al., FEMS Immunol. Med. Microbiol.658:1-7 (1996)!. The assay is performed in 96-well plates, and read at550 nm. NO generation is determined by quantitating nitrite, itsmetabolite, in L-arginine-supplemented culture medium of 10⁶ cellsincubated for 2 hours with bacteria. M. Schneemann et al., J. Infect.Dis. 167:1358-1363 (1993)!. Medium is centrifuged to remove bacteria,and supernatants added to Griess reagent (0.05%N-1-naphthylethylenediamine dihydrochloride, 0.5% sulfanilamide, 2.5%phosphoric acid) and incubated in 96-well plates for 10 minutes;absorbance is read at 570/630 nm. The lysosomal enzyme β-glucuronidaseis quantitated in medium and cell lysates W. Hsueh et al., Exp. LungRes. 13:385-399 (1987)! using the reagent 4-methyl umbelliferylβ-D-glucuronide trihydrate; fluorescence is read at 375/455.

Statistical Analysis

Data were analyzed using The Statview II® statistical package (AbacusConcepts). Comparisons for survival data were made using the Chi-squareanalysis. All other data are expressed as mean±SEM. Comparisons betweentreatment means were carried out using a two-tailed Student's t-test orthe Wilcoxen rank sum test, as appropriate (i.e., depending on whetherdata is parametric or non-parametric). For comparisons of mean data fromthree or more experimental groups, ANOVA and subsequent application ofthe Newman-Keuls test is used. The criterion for significance wasp≦0.05.

EXAMPLE 1 Survival Following Intratracheal Klebsiella Challenge In 5-LOKnockout Mice and Wild Type Mice

Intratracheal instillation of K. pneumoniae in mice is known to cause areproducible pneumonia characterized by acute pulmonary inflammationthat, depending on the inoculum, either resolves or results in death. G.Rosen et al., FASEB J. 9:200-209 (1995)!. To assess the role of 5-LOproducts in pulmonary host defense, this example compares the survivalof 5-LO knockout and wild type mice.

Profiles of Eicosanoids

FIGS. 2A and B depict RP-HPLC profiles of radioactive eicosanoidsreleased by prelabeled alveolar macrophages obtained from wild type mice(FIG. 2A) and 5-LO knockout mice (FIG. 2B). The profiles were obtainedby prelabeling 10⁶ alveolar macrophages overnight with ³ H!arachidonicacid. The alveolar macrophages were then washed and stimulated for 30minutes with 1 μM A23187. The medium was subjected to lipid extractionand radiolabeled eicosanoids separated by reverse-phase HPLC. Peaks wereidentified on the basis of co-elution with authentic standards. Ascompared to cells from wild type control animals (FIG. 2A), alveolarmacrophages from KO animals produced no leukotrienes or 5-HETE, asexpected. Moreover, there is no increased production of prostaglandinsfrom a possible "shunting" of acrachidonic acid. This indicates that anyreduction in antimicrobial defense in these animals is likelyattributable to their deficiency of pro-inflammatory leukotrienes,rather than to an overproduction of anti-inflammatory prostaglandin E₂.

Mouse Survival

For this experiment, 5-LO knockout mice and strain-matched (129/SvEv)wild type mice (ten animals per group) were inoculated intratracheallywith 50 CFU of bacteria, and survival was monitored over a 12-dayperiod. FIG. 3 graphically depicts the effect of K. pneumoniae challengeon survival in 5-LO knockout mice (solid circles) and wild type mice(open squares) (^(*) p<0.05 vs. WT). As the results in FIG. 3 indicate,administration of 50 CFU of bacteria led to 60% mortality in wild typemice within 8 days, with no subsequent deaths thereafter. In contrast,all of the knockout mice died in response to this same challenge, withall deaths occurring by day 10. Moreover, deaths in the knockout groupoccurred earlier than in the wild type animals.

These results indicate that the metabolic products of 5-LO play animportant role in the protective host response in this model ofpneumonia. The importance of early events following bacterial challengeis indicated by the fact that the survival curves in FIG. 3 diverge asearly as day 2.

EXAMPLE 2 Bacterial Clearance Following Intratracheal KlebsiellaChallenge In 5-LO Knockout Mice and Wild Type Mice

As set forth in the preceding example, early events following bacterialchallenge (i.e., approximately two-days post-challenge) are important.This example further explores those results by assessing lung homogenateand plasma CFUs at 30 and 48 hours after K. pneumoniae administration.

Knockout mice and wild type mice were inoculated with 50 CFUintratracheally, and lung homogenate levels and plasma CFU values weredetermined 48 hours later. FIG. 4 graphically depicts the clearance ofK. pneumoniae from lung and plasma after challenge in 5-LO knockout mice(cross-hatched bars) and wild type mice (solid bars) (bars representmean±SE; n=5-19 animals; ^(*) p<0.05 vs. WT). As indicated by the datain FIG. 4, mean lung as well as plasma CFUs were almost two logs greaterin knockout mice than in wild type mice at 48 hours post-challenge.Furthermore, the proportion of knockout animals that developedbacteremia at this time point (15/19) was significantly greater thanthat of wild type mice (10/19). In an additional group of animalsstudied at 30 hours post-challenge, 66% of knockout mice were bacteremic(average plasma CFU of 1.06×10⁵), while no wild type mice had bacteriain their plasma at this time point (data not shown).

These data confirm the importance of an intact leukotriene-generatingsystem for the early containment of a pulmonary challenge with K.pneumoniae.

EXAMPLE 3 Effect Of Leukotriene Deficiency And Exogenous Leukotrienes OnAlveolar Macrophage Antibacterial Functions In Vitro

The experiments of this example assess the ability of the alveolarmacrophages themselves, the first line of cellular defense, tophagocytose and kill K. pneumoniae in vitro and the effect ofadministering exogenous leukotrienes on alveolar macrophageantibacterial functions in vitro.

Phagocytic and Bactericidal Activities of Alveolar Macrophages from 5-LOKnockout and Wild Type Mice

Alveolar macrophages were purified by adherence of bronchoalveolarlavage cells lavaged from uninfected knockout and wild type animals, andpreincubated for 5 minutes with 5% K. pneumoniae-specific immune serum(as a source of both complement and specific opsonizing antibody) priorto assays. Cultured alveolar macrophages from either group of mice wereincubated in the presence of specific serum with K. pneumoniae for 1hour and then washed, after which monolayers were either stained withDiff-Quik (Difco) and intracellular organisms enumerated, or lysed andbacterial CFUs in lysates determined following overnight culture.Phagocytic index and intracellular killing were calculated as detailedabove in General Methods.

FIG. 5 graphically depicts phagocytic and bactericidal activities inalveolar macrophages isolated from 5-LO knockout mice (cross-hatchedbars) and wild type mice (solid bars); in FIG. 5, each value representsthe mean±SEM of 6 replicate cultures (^(*) p<0.05 vs. WT). As indicatedby the data in FIG. 5, alveolar macrophages from 5-LO knockout micedemonstrated significant decreases in their abilities to both ingest andkill K. pneumoniae when compared to cells from wild type mice. Sincekilling of microbes depends on their prior ingestion, the magnitude ofthe host defense defect in alveolar macrophages from knockout animalsreflects the arithmetic product of these two individual defects andamounts to approximately a 60% reduction in microbial killing under theconditions employed. Though exogenous LTB₄ has previously been reportedto enhance Gram-negative bacterial killing by macrophages in vitro andbacterial clearance in vivo T. Demitsu et al., Int. J. Immunopharmac.11:801-808 (1989)!, the data set forth above indicate an important rolefor endogenous 5-LO products in these same processes in vivo and invitro.

Effect of Exogenous LTB₄ on Bacterial Phagocytic Activity of AlveolarMacrophages from 5-LO Knockout and Wild Type Mice

Further experiments were performed to determine whether defectivephagocytosis of K. pneumoniae in alveolar macrophages from 5-LO knockoutanimals could be overcome by the addition of exogenous LTB₄. Thisparticular leukotriene was selected because, as previously indicated,its leukocyte-activating properties have been well-characterized.

Cultured alveolar macrophages from knockout mice were incubated in thepresence of specific serum for 1 hour with K. pneumoniae alone or in thepresence of varying doses of LTB₄. Phagocytic index was calculated asdescribed in the General Methods. FIG. 6 graphically depicts the effectof exogenous LTB₄ (none, 0.1 nM, and 5 nM LTB₄ added) on bacterialphagocytic activity in alveolar macrophages from 5-LO KO mice; eachvalue represents the mean from triplicate cultures. As shown in FIG. 6,LTB₄ dose-dependently enhanced the phagocytic index in knockout alveolarmacrophages, with an index approximately three times the baseline levelat a concentration of 5 nM. Though not required in order to practice thepresent invention, it is believed that neutrophils manifest similarfunctional defects in phagocytosis and killing which could contribute tothe sensitivity to bacterial pneumonia seen in knockout mice in vivo.

The effects of exogenous LTB₄ on phagocytosis by neutrophils from 5-LOknockout mice were also examined. Glycogen-elicited neutrophils wereobtained from the peritoneal cavity of knockout mice, and phagocytosisof K. pneumoniae over a one hour time period was evaluated in thepresence and absence of exogenous LTB₄ (1 nM); under thesecircumstances, phagocytic index was 27±8 and 45±4, respectively (datanot shown). These results with exogenous LTB₄ are important in severalrespects. First, they indicate that the phagocytic defect in these cellsis actually related to the deficiency of 5-LO, and is not coincidental.Second, the fact that addition of exogenous leukotriene could overcomethe lack of 5-LO indicates that the functional defect in these cells wascausally related to their endogenous leukotriene deficiency; thisfinding is contrary to the findings of other researchers who found thatfunctional defects in leukocytes caused by 5-LO inhibitors could not beovercome by addition of exogenous leukotrienes. See, e.g., N. Hubbardand K. Erickson, Mol. Immunol. 160:115-122 (1995)!. Third, the rapidityof the enhancement of phagocytic capacity produced by the addition ofexogenous leukotriene indicates that this effect might be reproduced bypulmonary delivery of this lipid in vivo; such a rapid increase inbacterial clearance has been observed upon injection of LTB₄ into theperitoneum in vivo. T. Demitsu et al., Int. J. Immunopharmac. 11:801-808(1989)!.

EXAMPLE 4 Inflammatory Cells And Mediators Following IntratrachealKlebsiella Challenge In 5-LO Knockout Mice and Wild Type Mice

This example evaluates the mechanisms responsible for the enhancedsusceptibility of knockout mice to Klebsiella pneumonia. Of course, itis to be understood that an understanding of the mechanisms is notrequired in order to practice the present invention.

Wild type mice were injected intratracheally with either 50 CFU ofbacteria (Klebsiella pneumoniae) or saline diluent alone. Two dayslater, lungs were harvested and homogenized. The homogenates weresubjected to lipid extraction, and immunoreactive leukotrienes B₄ and C₄were quantitated. FIG. 7 graphically depicts lung homogenate levels ofLTB₄ (hatched bars) and LTC₄ (solid bars) after challenge with either K.pneumoniae or saline (values represent mean±SEM; n=5 animals; ^(*)p<0.05 vs. saline).

As the results in FIG. 7 illustrate, both leukotriene B₄ and C₄ levelswere elevated in the lung homogenates of wild type mice 48 hours afterchallenge with bacteria as compared to saline. As LTB₄ is a potentneutrophil chemotaxin in mice and neutrophil recruitment is consideredan essential component of bacterial clearance, the presence of highlevels of LTB₄ in lungs of bacteria-challenged wild type animalsindicates that the enhanced susceptibility to pneumonia in knockoutanimals might reflect a reduced capacity to recruit neutrophils to theinfected organ. In order to evaluate that possibility, direct counts ofbronchoalveolar lavage neutrophils from cytospins (FIG. 8) wereperformed and lung homogenate MPO activity was spectrophotometricallyassayed (not shown) M. Greenberger et al., J. Immunol. 155:722-729(1995)!. Knockout and wild type mice were inoculated intratracheallywith either 50 CFU of bacteria or saline diluent alone. Two days later,lung lavage was performed and the total neutrophil count was determined.FIG. 8 graphically depicts the effect of K. pneumoniae challenge onlavage neutrophilia in 5-LO knockout (cross-hatched bars) and wild type(solid bars) mice (values represent mean±SEM; n=3-12 animals; ^(*)p<0.05 vs. saline; ND, none detected).

Both techniques indicated that a significant degree of neutrophil influxoccurred at 48 hours in bacteria-challenged as compared tosaline-challenged wild type lungs. Surprisingly, however, knockout miceexhibited no less neutrophil influx following bacterial challenge thandid wild type mice.

Though the precise mechanism is not required to practice the presentinvention, experiments were performed to determine whether the intactcapacity for neutrophil recruitment in this murine model reflects acompensatory increase in the knockout animals to generate alternativechemotactic signals such as chemokines. An evaluation of antigenicMIP-1α, MIP-2, and JE (the murine homologue of monocyte chemotacticpeptide-1) levels in homogenates of Klebsiella-challenged lungs at thissame time point (i.e., 48 hours post-challenge) disclosed no significantdifferences between knockout and wild type mice (data not shown).Alternatively, while an understanding of the mechanism is not requiredin order to practice the present invention, it is possible that theknockout animals might exhibit increased generation of complementcomponents or increased responsiveness to chemokines or bacterialchemotaxins.

Although not directly chemotactic, both IL-12 and TNF have been shown toplay critical protective roles in this model of murine pneumonia.Additionally, TNF production is potentiated by leukotrienes in someexperimental systems. To examine the possibility that the enhancedsusceptibility of knockout mice to bacterial challenge might relate toan impaired ability to generate either of these cytokines, lunghomogenates were analyzed 48 hours after bacterial challenge. Again, nosignificant differences were found in antigenic IL-12 or TNF levelsbetween infected knockout and wild type mice (data not shown). Thus, theincreased lethality of pneumonia in 5-LO knockout mice does not reflectdiminished capacity to produce these proinflammatory cytokines.

EXAMPLE 5 Effect of Exogenous Leukotrienes on Alveolar MacrophageAntibacterial Functions In Vitro

As reported above, exogenous LTB₄ increased the phagocytic index of 5-LOknockout alveolar macrophages by approximately 300%, more than wouldhave been necessary to merely attain the control level manifested bywild type cells (approximately 50% increase). That result indicates thatthe leukotriene is exhibiting a pharmacological effect. The experimentsof this example further evaluate the effects of exogenous LTB₄ onphagocytic capacity of normal alveolar macrophages and examine theeffects of other 5-LO products besides LTB₄.

Alveolar macrophages from Wistar rats were adhered and then incubatedfor 1 hour with K. pneumoniae alone or in the presence of 1 nM ofseveral 5-LO metabolites (LTB₄, LTC₄, and 5-HETE). Phagocytic index wassubsequently determined as described above in the General Methods.

FIG. 9 graphically depicts the effect of the exogenous 5-LO metaboliteson bacterial phagocytic activity in normal rat alveolar macrophages.Each value in FIG. 9 represents the mean±SEM of 4 replicate cultures. Asthe data indicate, LTB₄ evoked an approximately 6-fold increase inphagocytic index in normal rat alveolar macrophages. The metabolite5-HETE had a similar, though less pronounced, effect. Interestingly,LTC₄ augmented phagocytosis to a degree similar to LTB₄. Althoughcysteinyl leukotrienes like LTC₄ have been observed to upregulatesurface FcR expression in macrophages, increased phagocytic capacity hasnot been noted previously. These results indicate that the exogenousleukotrienes as a group appear to have a marked pharmacologic effect onnormal alveolar macrophage function.

A related experiment was also performed to determine if the ability ofexogenous LTB₄ to enhance bacterial phagocytosis is mediated by itsinteraction with LTB₄ receptors. This experiment was based on the factthat pretreatment with LTB₄ desensitizes cells to subsequent LTB₄responsiveness; though an understanding of the mechanism of this effectis not required to practice the present invention, the desensitizationis believed to occur by down-regulating receptor expression or coupling.For this experiment, cells were pretreated with LTB₄ (1 nM) for 1 hour,washed, and incubated with bacteria plus LTB₄.

The results, graphically depicted by the bar labelled "LTB₄ →LTB₄ " inFIG. 9, indicate that pre-treatment with LTB₄ almost completelyabrogated the ability of this same dose of LTB₄ (1 nM) to augmentphagocytosis of K. pneumoniae when added simultaneously with bacteria.The findings indicate that LTB₄ receptors are involved in theenhancement of alveolar macrophage phagocytosis induced by LTB₄.

EXAMPLE 6 Effect Of Intratracheal LTB₄ Administration On PulmonaryBacterial Clearance By Knockout Mice

Because it was found that 5-LO knockout mice displayed reduced pulmonaryclearance of K. pneumoniae in vivo, and exogenous leukotrienes were ableto overcome the in vitro phagocytic defect observed in alveolarmacrophages from knockout mice, an experiment was performed to evaluatethe effect of intrapulmonary administration of leukotriene on bacterialclearance in vivo.

LTB₄ was administered together with the intratracheal inoculum of K.pneumoniae (50 CFU). A dose of 6 ng of LTB₄ intratracheally per animalwas chosen for two reasons. First, other researchers previously foundthat this dose and route resulted in a brisk neutrophil influx 6 hoursafter administration in mice. N. Ahmed et al., Am J. Respir. Crit. CareMed. 153:1141-1147 (1996)!. Second, the present inventors previouslyfound (see FIG. 5) that approximately 7 ng of total LTB₄ could bemeasured in the homogenate of a pair of lungs from Klebsiella-challengedwild type mice. Three groups of animals were challenged intratracheallywith bacteria (n=4 animals per group): i) wild type mice, ii) 5-LOknockout mice, and iii) 5-LO knockout mice treated concomitantly withLTB₄. Following 24 hours of bacterial inoculation, lungs were harvestedand lung homogenate CFUs were determined.

FIG. 10 graphically depicts the effect of intratracheal administrationof LTB₄ on defective bacterial clearance of the lung in 5-LO knockoutmice (each value represents the mean±SEM). The data shown in FIG. 10confirm the previous finding (FIG. 4) that knockout mice hadapproximately 100-fold more organisms in their lungs than did wild typeanimals; it should be noted that the absolute CFUs in this experimentwere less because analysis was performed at 24 hours after inoculationrather than 48 hours. Importantly, the single intratracheal dose of LTB₄administered concomitantly with the bacterial inoculum reduced the lungCFU by approximately 10-fold in knockout mice. The results indicate thatexogenous LTB₄ is capable of augmenting pulmonary clearance of K.pneumoniae in these leukotriene-deficient mice. Moreover, they indicatethat leukotrienes should be effective therapeutic agents in the settingof Gram-negative pneumonia.

EXAMPLE 7 The Roles And Mechanisms Of Action Of 5-LO Products In TheHost Response To K. pneumoniae

The examples described above employing intratracheal Klebsiellachallenge in 5-LO knockout mice demonstrate that the enzyme plays an invivo role in pulmonary antibacterial host defense. The experiments ofthis example are directed at ascertaining the roles and mechanisms ofaction of 5-LO products in the host response to K. pneumoniae usingknockout mice as well as mice treated with pharmacological agents whichinhibit leukotriene synthesis or actions. More specifically, theexperiments of this example are directed at discerning the role of LTB₄vs. cysteinyl leukotrienes by comparing the effects of a variety ofpharmacologic agents, including those which target both classes ofleukotrienes (5-LO inhibitor), those which target only LTB₄ (LTB₄receptor antagonist), and those which target only cysteinyl leukotrienes(cysteinyl leukotriene receptor antagonists).

The murine model involving intratracheal challenge of mice with 50 CFUof K. pneumoniae is utilized in the experiments of this example. Inorder to pharmacologically interfere with leukotriene synthesis oraction, wild type mice are treated with various long-acting agents (setforth below) by the oral (gavage) route, with daily dosing commencingthe morning of the day before the administration of bacteria. In allcases, the specificity of the agents to be used has been established,and the selection of doses and dosing regimens is guided by publishedexperience in rodents. On the basis of preliminary dose-responseexperiments employing three doses per agent and n=4 animals per dose, asingle maximally effective dose of each drug is determined fromassessments made at 24 hours after initiation of treatment.

The specific agents and preliminary dose ranges which are tested includethe following: i) the 5-LO inhibitor A-79175 (Abbott) in a 1-3 mg/kgdose; this is a competitive enzyme inhibitor that is a more potent andlonger-acting congener of Zileuton® with demonstrated efficacy in miceas a once-daily oral agent; ii) the LTB₄ antagonist CP-105,696 (Pfizer)in a 1-10 mg/kg dose; this compound has inhibited collagen-inducedarthritis in mice when administered in a once-daily oral dose; and iii)the LTD₄ antagonist MK-571 (Merck) in a 0.1-1 mg/kg dose; this compoundhas effectively inhibited antigen-induced bronchoconstriction whenadministered orally to rats.

Once the optimal dose of each agent is defined, survival and bacterialclearance experiments are performed separately, each involving K.pneumoniae challenge of the following five groups of mice (n=10 pergroup): i) wild type mice treated with vehicle; ii) wild type micetreated with the 5-LO inhibitor; iii) wild type mice treated with theLTB₄ antagonist; iv) wild type mice treated with the cysteinylleukotriene antagonist; and v) 5-LO knockout mice treated with vehicle.In vivo efficacy is judged by the following criteria. 5-LO inhibition isevaluated by quantitating pulmonary production of LTB₄ (quantitated inlung lavage fluid) following intratracheal instillation of ionophoreA23187 in drug-treated animals. W. Smith et al., J. Pharmacol. Exp.Ther. 275:1332-1338 (1995)!. LTB₄ antagonism is assessed by quantitatingthe ex vivo LTB₄ -stimulated upregulation of CR3 expression onneutrophils in whole blood obtained from drug-treated animals. Cysteinylleukotriene receptor antagonism is assessed by quantitating Evans bluedye extravasation following intradermal administration of LTD₄. J.Drazen et al., Proc. Natl. Acad. Sci USA 77:4354-4358 (1980)!.

Animal survival is monitored until death or until day 14. For bacterialclearance, bacterial CFU is determined in whole lung homogenates andplasma obtained from animals sacrificed at both 1 day and 3 dayspost-Klebsiella challenge. Finally, lung neutrophil influx is initiallyassessed by MPO activity of whole lung homogenates from the same animalsused for CFU determinations above; if MPO assays suggest that activedrug treatment results in a reduction in neutrophil influx, anadditional experiment is carried out (since lavage and homogenizationcannot be performed in the same animal) in which such an effect isverified by bronchoalveolar lavage cell counts and differentials ondrug- vs. vehicle-treated animals.

It should be noted that determining the relative contribution to hostdefense of endogenously synthesized LTB₄ versus LTC₄ allows i) thedesign of therapeutic studies employing administration of exogenousleukotrienes and ii) the assessment of possible risks to infectionsusceptibility of, for example, 5-LO inhibitors (which inhibit synthesisof LTB₄ and cysteinyl leukotrienes in parallel) and cysteinylleukotriene receptor antagonists (which selectively inhibit the actionsof cysteinyl leukotrienes without affecting those of LTB₄).

If direct inhibition of 5-LO impairs survival and bacterial clearance inthis murine pneumonia model in a manner similar to 5-LO deficiency, therelative roles of endogenous LTB₄ vs. cysteinyl leukotrienes areassessed by the application of receptor antagonists which selectivelyblock the actions of these two groups of mediators. Although LTB₄ is the5-LO metabolite most generally implicated in leukocyte-dependentinflammatory reactions, previously generated phagocytosis data suggestthat cysteinyl leukotrienes might have comparable enhancing effects.Conversely, if anti-leukotriene agents do not reproduce the effects ofthe 5-LO gene deficiency, it will suggest that 5-LO enhancesantibacterial defense by a mechanism independent of its catalyticactivity. If pharmacologic inhibitors/antagonists do impair hostdefense, a determination is made as to whether the relevant mechanism isindependent of impairment of neutrophil recruitment to the lung.Finally, the possibility that anti-leukotriene therapy augments the hostresponse to Klebsiella pneumonia (i.e., leukotrienes both enhance andimpair the host response) is considered. Indeed, results indicating thateach of these opposing effects predominates at different phases of theresponse may warrant the use of the pharmacologic agents employed atspecific intervals.

EXAMPLE 8 The Kinetics, Profile, and Cellular Sources of LeukotrienesProduced in the Murine Lung During the Course of Klebsiella Pneumonia

Experiments described in previous examples (see, e.g., FIG. 3) indicatedthat both LTB₄ and LTC₄ are present at high levels in lung homogenates48 hours after bacterial challenge. The experiments in this example aredirected at determining which leukotrienes are produced in the lung atdifferent time points following K. pneumoniae challenge and which celltypes are responsible. The initial experimental objective is toquantitate leukotrienes in lung homogenates and bronchoalveolar lavagefluid from mice at various time points post-Klebsiella challenge. On thebasis of these data, time points are selected for further studiesdesigned to determine the cellular sources of leukotrienes through i)immunohistochemical staining in order to identify cells exhibiting anintracellular distribution of 5-LO associated with enzyme activation,and ii) measuring constitutive leukotriene production by cells isolatedfrom pneumonic lungs.

Initially, 129/SvEv wild type mice are inoculated intratracheally witheither saline or with 50 CFU of K. pneumoniae, and lungs are harvestedat 8 hours and 1, 2, 3, 5, and 7 days post-inoculation. For each ofthese time points following saline or bacteria innoculation, whole lunghomogenates are prepared (n=5 animals per group) and both LTB₄ and LTC₄are quantitated in homogenates by immunoassay. In other animals (n=3),lung sections are prepared for immunohistochemistry (see below). Inparallel, an identical experiment is conducted in which lung lavage isperformed; at each time point (n=5 animals per group), bronchoalveolarlavage cytospins are prepared and levels of leukotrienes are determinedin cell-free lavage fluid. Levels of LTB₄ and LTC₄ in lavage fluid andin lung homogenates are correlated with each other and with the degreeof neutrophil influx (assessed from MPO activity in homogenates and cellcounts and differentials from bronchoalveolar lavage fluid cytospins).

The cellular sources of leukotriene production in the lung is determinedon the 8 hour, 1 day, and 3 day time points and other time pointsidentified by the above kinetic analysis indicating maximal levels ofleukotrienes B₄ or C₄. Immunohistochemical staining for 5-LO isperformed on lung sections along with bronchoalveolar lavage cytospinpreparations from both Klebsiella- and saline-challenged mice in orderto determine whether it is the alveolar macrophages, neutrophils, orboth cell types which demonstrate an intracellular distribution of 5-LOcharacteristic of enzyme activation (i.e., staining concentrated at thenuclear envelope). Determining 5-LO activation in lung tissue in situ bythis method has the advantage that it does not require cell isolation orculture, obviating concerns about the potential artifacts which might beintroduced by those procedures. Of note, this approach has been used inidiopathic pulmonary fibrosis to demonstrate that alveolar macrophagesisolated by bronchoalveolar lavage from patients with idiopathicpulmonary fibrosis constitutively overproduce leukotrienes when placedinto culture, even in the absence of an exogenous agonist. J. Wilborn etal., J. Clin. Invest. 97:1827-1836 (1996)!.

As described above, there is overproduction of leukotrienes in lungtissue at 2 days following bacterial challenge. In order to determinewhether bronchoalveolar cells from bacteria-inoculated animals continueto elaborate leukotrienes after being placed into culture in a mannerwhich reflects their prior generation in vivo, unfractionatedbronchoalveolar lavage cells (10⁶ cells) are obtained at the time pointsmentioned above, plated in culture dishes, and cumulative production ofleukotrienes B₄ and C₄ are assessed by immunoassay of culture mediumfollowing overnight (approximately 16 hours) culture; bronchoalveolarlavage cells from control animals are studied for comparison (n=5animals per treatment per time point). Following overnight culture,adherent cell differentials are determined by Wright's and esterasestaining.

It should be noted that studying mixed cell populations should notcreate difficulty in attributing leukotriene generation to a particularcell type at the 8 hour time point because there is a relatively purepopulation of alveolar macrophages at the time. Furthermore, alveolarmacrophages and neutrophils synthesize unique profiles of leukotrieneproducts; thus, alveolar macrophages produce primarily LTC₄ (FIG. 2A)while neutrophils synthesize primarily LTB₄. When interpreted inconjunction with the immunohistochemical data, the profile ofleukotrienes elaborated by cultured bronchoalveolar lavage cellsprovides strong evidence for the involvement of each cell type. Finally,studying mixed lavage cells allows potential neutrophil-alveolarmacrophage interactions in leukotriene synthesis to take place, as theyinevitably do in vivo.

Knowledge of the kinetics of endogenous production of LTB₄ vs. LTC₄ ishelpful in several important respects. First, it provides guidance indesigning the "therapeutic" experiments (described below) involvingpulmonary administration of exogenous leukotrienes. Second, determiningthe contributions of alveolar macrophages and neutrophils as sources forthe production of these mediators provides basic information about thebiology of the host response. Finally, knowledge of the appropriatecellular sources of leukotrienes in the setting of bacterial pneumoniahas potential diagnostic utility in that documenting deficientleukotriene production may help to identify patients who may becandidates for exogenous pulmonary leukotriene supplementation in orderto augment innate immunity.

EXAMPLE 9 The Molecular Mechanisms by Which Specific 5-LO ProductsAugment Phagocytosis and Killing of K. Pneumoniae in AlveolarMacrophages and Neutrophils

The experiments of this example elucidate the molecular mechanisms bywhich specific 5-LO metabolites enhance phagocytosis and killing. Morespecifically, the experiments of this example involve adding differentlipids to alveolar macrophages and elicited neutrophils obtained bothfrom knockout mice and from wild type mice in order to compare themagnitude of effects and mechanisms of action for different 5-LOproducts in both cell types. These experiments provide a means of i)further evaluating the therapeutic utility of leukotrienes, and ii)evaluating the utility of particular molecular and/or biochemicalmarkers as endpoints to be examined in the in vivo leukotriene treatmentstudies described below in Example 10.

As described in detail hereafter, initial experiments characterize theeffects of in vitro incubation with exogenous 5-LO products on the crudeendpoints of phagocytosis and killing of K. pneumoniae. Though anunderstanding of the molecular mechanisms is not required in order topractice the present invention, because the molecular mechanismsmediating bacterial phagocytosis and killing are quite similar inneutrophils and macrophages and strong evidence exists implicating rolesfor the 5-LO pathway in functions of both cell types, studies areperformed in both alveolar macrophages and glycogen-elicited peritonealneutrophils from mice (purity of both populations exceeds 90%). Of note,neutrophil recruitment to the peritoneum following glycogen elicitationhas been shown not to be impaired in 5-LO knockout mice. X. Chen et al.,Nature 372:179-182 (1994)!. Elicited neutrophils are studied instead ofperipheral blood neutrophils because of the possibility that the processof recruitment and/or residence in an inflammatory milieu itself alterscellular phenotype. In addition, cells obtained from both wild type andknockout mice are studied.

Specifically, 10⁵ cells are coincubated for 1 hour with bacteria andlipids in the presence of 5% immune serum, and phagocytic index andbactericidal activity are assessed as described above under GeneralMethods. In each experiment, a vehicle control is included. Theexogenous 5-LO metabolites to be studied (all at 10⁻¹¹ -10⁻⁷ M) are i)LTB₄, ii) LTC₄ ; and iii) 5-HETE. Combinations of these lipids are alsoevaluated.

For 5-LO products that have stimulatory effects on phagocytosis orkilling, the ability of specific receptor antagonists (described inExample 7) to abrogate these effects are also tested. All studies arecarried out with both neutrophils and alveolar macrophages in order toensure that instances in which a given compound exerts different effectson phagocytosis in the two cell types and exerts similar effects (thoughmediated by different mechanisms) in the two cell types are identified.Once a molecular mechanism for an effect on phagocytosis is identified(e.g., by LTB₄), the ability of the opposing compound (i.e., the LTB₄receptor antagonist) to modulate that same molecular event in anopposite fashion is examined.

The mechanistic endpoints for study are as follows: i) surfaceexpression of receptors necessary for binding/ingestion of K. pneumoniae(assessed by flow cytometry), including FcRII/FcRIII and CR3; ii) actinmicrofilament assembly (assessed by immunofluorescent staining and flowcytometry), necessary for particle engulfment; and iii)phagosome-lysosome fusion (assessed by acridine orange staining),necessary to bring the microbe in contact with the bactericidal arsenal.The General Methods describes the procedures for each of theseassessments.

Bactericidal mechanisms are examined in a manner similar to thatdescribed for phagocytosis. Again, while an understanding of themolecular mechanisms is not required in order to practice the presentinvention, subsequent experiments are performed to address the molecularmechanism(s) that are responsible for 5-LO metabolites that augmentkilling of K. pneumoniae. Moreover, the ability of antagonists to blockthe positive effects of lipids on these mechanistic events areevaluated, and alveolar macrophages and elicited neutrophils are bothstudied. Three bactericidal mechanisms are examined (as described in theGeneral Methods section). First, extracellular generation of O₂ - isassessed by the superoxide dismutase-inhibitable reduction offerricytochrome C. L. Laichalk et al., FEMS Immunol. Med. Microbiol.658:1-7 (1996)!. Because bacteria may not represent a sufficientlystrong stimulus for extracellular release of oxygen metabolites, theeffects of leukotrienes on this endpoint are also assessed using phorbolmyristate acetate as the stimulus for O₂ -production. Second, productionof NO is determined by quantitating nitrite in culture medium using theGriess reagent. Third, release of a lysosomal enzyme, β-glucuronidase,is determined spectrophotometrically. W. Hsueh et al., Exp. Lung Res.13:385-399 (1987)!.

Following characterization of the effects of exogenous leukotrienes onthese molecular mechanisms in knockout as well as wild type cells, it isdetermined whether specific antagonism of these same leukotrienesproduced endogenously has the same effects. As in Example 7, the LTB₄antagonist CP-105,696 and the cysteinyl leukotriene antagonist MK-571(both at 10⁻⁹ -10⁻⁶ M) are used. They are added to wild type cells priorto addition of K. pneumoniae, and phagocytosis, killing, and relevantmolecular mechanisms are then evaluated as described above.

If LTB₄ and LTC₄ are shown to exert their effects via differentmechanisms, the combination of the two might activate antibacterialfunctions in a manner that is additive or synergistic. Such a findinghas important implications for possible therapeutic use of leukotrienesin the in vivo studies described in Example 10.

EXAMPLE 10

The Effects of Aerosolized or Intratracheal Leukotrienes Post-KlebsiellaChallenge on Bacterial Clearance and Survival in Both Wild Type and 5-LOKnockout Mice

The data obtained from the preceding examples provides, among otherthings, information regarding the time point in the host response atwhich the presence of particular leukotrienes is most critical. Theexperiments of this example use that information to rationally test thein vivo efficacy of exogenous leukotrienes, either singly or incombination, administered by different routes.

The initial experiments of this example involve animals whose endogenouscapacity for leukotriene generation is impaired because of 5-LO genedisruption. Subsequent experiments test the efficacy of intrapulmonaryleukotriene administration in wild type mice. Finally, in addition tothe clinically relevant endpoints of bacterial clearance and survival,the experiments of this example investigate the utility of profiling amolecular consequence of leukotriene action (e.g., CR3 expression) onlavaged cells as a possible surrogate for predicting diminished (withoutexogenous leukotrienes) or enhanced (with exogenous leukotrienes)bacterial clearance and survival.

"Early" Administration of Leukotrienes

Because of the data previously described (see FIG. 10) and becauseleukotriene-deficient animals are expected to manifest the greatestincrement in antimicrobial defense from administration of exogenousleukotrienes, 5-LO knockout mice are used for the first series ofstudies. Knockout mice are given 50 CFU of K. pneumoniae intratracheallytogether with LTB₄ in doses ranging from 1-20 ng per animal (6 ng wasthe dose utilized in the experiment corresponding to FIG. 10); a similardose range of LTC₄ is also tested. Lung and plasma bacterial CFUs aredetermined at 1 day, and the results of these experiments are used todetermine optimal doses of concomitantly administered LTB₄ and LTC₄.Next, the effects on in vivo bacterial clearance are definitivelyassessed from lung and plasma CFUs at both 1 and 3 days postinoculation, using n=10 knockout animals for each assessment time pointper treatment group, as follows: i) vehicle control (bacteria only) isassessed at 1 day; ii) vehicle control is assessed at 3 days; iii) LTB₄is assessed at 1 day; iv) LTB₄ is assessed at 3 days; v) LTC₄ isassessed at 1 day; vi) LTC₄ is assessed at 3 days; vii) LTB₄ +LTC₄ areassessed at 1 day; and viii) LTB₄ +LTC₄ are assessed at 3 days. Forfurther comparison, wild type animals inoculated with bacteria alone arestudied at both time points (groups ix) and x)). Because combinations ofoptimal doses of LTC₄ and LTB₄ might prove excessively pro-inflammatory,such combination therapy may require that the doses of each agent bescaled back.

The treatment regimen(s) that yield(s) the greatest improvement inbacterial clearance is then utilized in a survival experiment. Onceagain, knockout mice (n=10 animals per group) are inoculated with 50 CFUof K. pneumoniae alone or together with optimal doses of LTB₄, LTC₄, orboth leukotrienes; survival is monitored over 14 days. Wild type miceinoculated with bacteria alone serve as another comparison group.

"Late" Administration of Leukotrienes

Since prior experiments indicate that the effects of an intratrachealdose of leukotriene are rapid in onset (e.g., within 1 hour) butrelatively short-lived (e.g., less than 12 hours), then administeringleukotriene(s) together with the bacterial inoculum should augment thebacterial clearance potential of the alveolar macrophage. Alternatively,administering leukotriene(s) at a later time point is associated withother potential merits. For example, activation of the recruitedneutrophils might be accomplished if active compound is dispensed atapproximately 1-3 days post-inoculation. Moreover, an efficaciouspost-inoculation regimen is more readily applicable to treatment ofoverwhelming Gram-negative pneumonia in patients.

In light of the above, experiments are performed to define optimal timepoints (1, 2, and 3 days post-Klebsiella challenge) for "late"administration of LTB₄ and LTC₄. These are carried out in knockout miceand bacterial clearance (lung and plasma CFUs) are assessed at day 4;leukotriene-treated animals are then compared to no-leukotriene(vehicle) controls. Following determination of the best "late" timepoint, lung and plasma CFUs are determined 1 day thereafter in thefollowing groups: i) bacteria alone, ii) bacteria+LTB₄, iii)bacteria+LTC₄, and iv) bacteria +LTB₄ +LTC₄ (n=10 animals per group).For further comparison, wild type animals inoculated with bacteria aloneare studied. As described for the simultaneous treatment regimen, theoptimal late treatment regimen is next tested in knockout mice in a 14day survival study, with vehicle-treated knockout mice and wild typemice serving as comparison groups.

Simultaneous "Early" and "Late" Administration of Leukotrienes

Repeated or prolonged administration of leukotrienes may augmentantibacterial host defense to a greater degree than either early or lateadministration alone. As a result, two additional regimens areperformed. For both of these alternative regimens, 5-LO knockout miceare utilized, and bacterial clearance experiments are carried out firstand optimal regimens are subsequently tested in longer survivalexperiments. The first regimen entails early (e.g., with inoculation)and late (e.g., day 2) administration. The early and late 5-LOmetabolite can be selected independent of each other; in other words,LTB₄ can be utilized for one dose and LTC₄ for the other dose. Thesecond regimen entails continuous administration of leukotriene(s) byaerosol. To ensure dosing limited to the respiratory tract and to beable to precisely quantitate the dose administered, leukotrienes arenebulized and administered to mice via a nose-only exposure chamber.Selection of the metabolite and the treatment window (e.g., days 1-3) isbased on the results from the one-time dosing experiments.

Application to K. pneumoniae-challenged wild type mice

The experiments set forth above regarding leukotriene-deficient mice areapplied to K. pneumoniae-challenged wild type mice. 129/SvEv wild typemice are more susceptible to Klebsiella pneumonia than are many otherstrains, although not as susceptible as 5-LO knockout mice. These wildtype mice may therefore be more closely representative of patientssusceptible to Gram-negative pneumonia than are theleukotriene-deficient animals. Therefore, the optimal leukotrienetreatment strategy defined from studies in knockout mice is used in wildtype mice, with similar endpoints of bacterial clearance and survival.

The experiments disclosed in this example indicate the effects ofaerosolized and intratracheal administration of post-Klebsiellachallenge on bacterial clearance and survival in both wild type and 5-LOknockout mice. These experiments serve to provide information regardingthe in vivo administration of exogenous leukotrienes. The studiesdescribed involve treatment with leukotrienes B₄ and C₄ ; these wereselected because of their known actions and their potency. However, theuse of other 5-LO products, including 5-HETE and lipoxins iscontemplated by the present invention.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds, composition, methods, orprocedures shown and described, as modifications and equivalents will beapparent to one skilled in the art.

We claim:
 1. A method of enhancing antimicrobial defense, comprisingadministering an effective amount of a therapeutic composition to a hostsuspected of having a microbial infection, said composition comprising acysteinyl leukotriene.
 2. The method of claim 1, wherein said microbialinfection is bacterial pneumonia.
 3. The method of claim 1, wherein saidcysteinyl leukotriene is selected from the group consisting ofleukotriene C₄, leukotriene D₄ and leukotriene E₄.
 4. A method ofenhancing antimicrobial defense, comprising pulmonary administration ofan effective amount of a therapeutic composition to an immunocompromisedhost, said composition comprising leukotriene B₄.
 5. The method of claim4, wherein said pulmonary administration is by aerosolization of saidtherapeutic composition.
 6. The method of claim 1, further comprisingthe co-administration of an antibiotic to said host.
 7. The method ofclaim 1, wherein said host is an animal.
 8. The method of claim 1,wherein said host is a human.
 9. A method of treating a bacterialinfection, comprising pulmonary administration of an effective amount ofa therapeutic composition to a host suspected of having a bacterialinfection, said therapeutic composition comprising leukotriene B₄. 10.The method of claim 9, wherein said bacterial infection is bacterialpneumonia.
 11. The method of claim 9, wherein said pulmonaryadministration is by aerosolization of said therapeutic composition. 12.The method of claim 11, further comprising the co-administration of anantibiotic to said host.
 13. The method of claim 9, wherein said host isan animal.
 14. The method of claim 9, wherein said host is a human.